The invention relates to humanized antibodies that specifically bind to 4-1BB receptor proteins, preferably to human 4-1BB receptor protein. The humanized antibodies can be used as diagnostic reagents or can be formulated into pharmaceutical compositions for administration to a patient.
The immune system has tremendous diversity and because the repertoire of specificities expressed by the B- and T-cell populations is generated randomly, it is bound to include many which are specific for self components. Thus, the body must establish self-tolerance mechanisms to distinguish between self and non-self determinants so as to avoid autoreactivity. However, all mechanisms have a risk of breakdown. The self-recognition mechanisms are no exception, and a number of diseases have been identified in which there is autoimmunity due to copious production of autoantibodies and autoreactive T cells.
There are at least 30 diseases which are either caused by or related to autoimmunity. Examples of such diseases include rheumatoid arthritis, pemphigus vulgaris, glomerulonephritis, pernicious anemia, thyroiditis and systemic lupus erythematosus. In Korea, one person in one hundred suffers from rheumatoid arthritis.
The transplantation of tissues to replace diseased organs is now an important medical therapy. In most cases, adaptive immune responses to the grafted tissues are the major impediment to successful transplantation. When tissues containing nucleated cells are transplanted, T-cell responses to the highly polymorphic MHC (major histocompatibility complex) molecules almost always trigger a response against the grafted organ. Matching the MHC type of donor and recipient increases the success rate of grafts, but perfect matching is possible only when donor and recipient are related and, in these cases, genetic differences at other loci still trigger rejection.
The immune system may be manipulated or controlled to suppress unwanted immune responses in autoimmune disease and graft rejection. Currently, several different immunosuppressive agents have been used clinically. The examples are methotrexate, azathiopurine, cyclophosphamide, prednisone, cyclosporine A, FK506 (tacrolimus), anti-lymphocyte globulin (ALG) and anti-thymocyte globulin (ATG). Very recently, antibodies by virtue of their exquisite specificity have been utilized for the therapeutic inhibition of specific immune responses. The target molecules for these antibodies can be divided into two groups. The first group includes molecules that are expressed on the surface of lymphocytes, such as CD3, CD4, IL-2R, CDw52 and ICAM-1. The other groups are mainly cytokines such as TNF-xcex1 and IL-6. Some of the antibodies are effective and are being sold as pharmaceutical products.
However, the presently developed immunosuppressants have a common problem in that cells that are not related to immune response or normal cells are all affected by the drugs. This causes serious side effects that cannot be avoided. Therefore, an immunosuppressant is desired that is specific for activated immune cells, has excellent immunosuppressive activity, and has no adverse side effect.
Although murine monoclonal antibodies are extensively used as diagnostic agents, their utility as therapeutics has been proven in only a few cases. The limited application is attributed to three major reasons. First, the repeated administration of murine monoclonal antibodies to humans usually elicits human immune responses against these molecules. The human anti-mouse antibody (HAMA) responses are directed to two different domains. The responses against the variable region are so called anti-idiotype responses which could block the antigen binding activity of murine antibodies. The responses against the constant region represent anti-isotype responses, which block the effector function of antibodies. The HAMA responses not only block the functions of newly administered antibodies but also result in formation of immune complexes with the murine antibodies, which cause some side effects and could reduce the half life of the antibody. Second, the half-life of murine antibodies even in the absence of immune complex formation is much shorter than that of human antibodies in vivo. Third, the effector functions through the Fc region of murine antibodies are weak or non-existent compared to those of human antibodies. All of the factors described above reduce the efficacy of murine monoclonal antibodies and are common problems related to human immunotherapy based on xenogeneically derived monoclonal antibodies.
To overcome the intrinsic undesirable properties of murine monoclonal antibodies, recombinant murine antibodies engineered to incorporate regions of human antibodies, so called xe2x80x9chumanized antibodiesxe2x80x9d, have been developed. This alternative strategy was adopted as is was very difficult to generate human antibodies directed to human antigens, such as cell surface molecules, due to tolerance of the immune system against self-antigens. A humanized antibody contains complementarity determining region (CDR) regions and a few other amino acid of a murine antibody and the rest of the structure is derived from a human antibody.
4-1BB is expressed on the surface of activated T-cells as a type of accessory molecule (Kwon et al., Proc. Natl. Acad. Sci. USA 86:1963 (1989); Pollok et al., J. Immunol. 151:771 (1993)), and is a membrane protein related to tumor necrosis factor receptor (TNFR) (Malett et al., Immunol. Today 12:220 (1991)). 4-1BB has a molecular weight of 55 kDa, and is found as a homodimer. In addition, 4-1BB binds to the protein kinase p561ck inside the cell. It has been suggested that 4-1BB mediates a signal transduction pathway from outside of the cell to inside (Kim et al., J. Immunol. 151:1255 (1993)).
A human 4-1BB gene was isolated from a CDNA library made from activated human peripheral T-cell mRNA (Goodwin et al., Eur. J. Immunol. 23:2631 (1993)). The amino acid sequence of human 4-1BB shows 60% homology to mouse 4-1BB (Kwon et al., Proc. Natl. Acad. Sci. USA 86:1963 (1989)), which indicates that the sequences are highly conserved. Analysis of the amino acid sequence of 4-1BB indicates that it belongs to the nerve growth factor superfamily, along with CD40, CD27, TNFR-I, TNFR-II, Fas, and CD30 (Alderson et al., Eur. J. Immunol. 24:2219 (1994). When a monoclonal antibody is bound to 4-1BB expressed on the surface of mouse T-cells, anti-CD3 T-cell activation is increased many fold (Pollok et al., J. Immunol. 150:771 (1993)).
4-1BB binds to a high affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells (Pollok et al., J. Immunol. 150:771 (1993); Schwarz et al., Blood 85:1043 (1995)). The interaction of 4-1BB and its ligand provides a costimulatory signal leading to T cell activation and growth (Goodwin et al., Eur. J. Immunol. 23:2631 (1993); Alderson et al., Eur. J. Immunol. 24:2219 (1994); Hurtado et al., J. Immunol. 155:3360 (1995); Pollock et al., Eur. J. Immunol. 25:488 (1995); DeBenedette et al., J. Exp. Med. 181:985 (1995). These observations suggest an important role for 4-1BB in the regulation of T cell-mediated immune responses (Ignacio et al., Nature Med. 3:682 (1997)).
The inventors have previously constructed a hybridoma producing a mouse monoclonal antibody that is specific for human 4-1BB (h4-1BB) expressed on the surface of activated T-cells (Korean patent laid-open no. 96-37064). As a result of the search by the inventors for an immunosuppressant specific for activated T-lymphocytes and that also has no adverse side effect, the inventors have constructed a humanized monoclonal antibody from a mouse monoclonal antibody to 4-1BB that is expressed only in activated T-lymphocytes (Korean patent laid-open no. 96-37064). The humanized monoclonal antibody has high affinity for 4-1BB. Administering the humanized anti-4-1BB monoclonal antibody to non-human primates does not elicit an anti-antibody response. Rather, it elicits strong immunosuppressive activity.
One object of the present invention is to provide a humanized monoclonal antibody that specifically binds 4-1BB, especially human 4-1BB (h4-1BB) that has a high affinity for 4-1BB (h4-1BB). The humanized antibodies of the present invention have high affinity for human 4-1BB, and bear sequence similarity to human antibodies. Because the 4-1BB receptor protein that is specifically bound by the antibodies of the invention appears to be involved in activation of the immune response, the product can be used effectively to treat autoimmune diseases or it can be used as an immunosuppressant to prevent graft rejection, Because the antibodies of the present invention closely resemble human antibodies, they can be administered to a human patient without any negative side-effect, such as a human anti-mouse antibody response.
Another object of the present invention is to provide a pharmaceutical composition comprising the humanized anti-h4-1BB monoclonal antibody. The pharmaceutical composition is useful for treating autoimmune diseases or acting as an immunosuppressant to prevent graft rejection. As rheumatoid arthritis is thought to be caused by inappropriate activity of 4-1BB receptor, the composition is especially useful for treating rheumatoid arthritis.
Another object of the invention is to provide a diagnostic composition for diagnosis of immune dysfunctions related to over- or underactivity of 4-1BB receptor protein.